Oncolytic Viruses as Phenotyping Agents for Neoplasms

ABSTRACT

The present invention provides a method of diagnosing neoplasms having a particular phenotype by using oncolytic viruses that selectively replicate in neoplasms having the particular phenotype. For example, reovirus does not replicate in normal cells. However, reovirus selectively replicate in cells with an activated ras pathway, which leads to death of these cells. Therefore, a cell which becomes neoplastic due to, at least in part, elevated ras pathway activities can be diagnosed by its susceptibility to reovirus replication. This invention can further be applied, using other oncolytic viruses, to the diagnosis and/or treatment of other tumors, such as interferon-sensitive tumors, p53-deficient tumors and Rb-deficient tumors. Kits useful in the diagnosis or treatment disclosed herein are also provided.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationsSer. No. 60/392,031, filed Jun. 28, 2002; and Ser. No. 60/443,188, filedJan. 29, 2003. The entire disclosure of each of these prior applicationsis hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods of detecting the underlying cause oftumors, particularly the use of reovirus in the diagnosis ofras-activated tumors. In addition, other oncolytic viruses withdifferent selectivities can also be used in the diagnosis of particulartumor types.

REFERENCES

U.S. Pat. No. 6,136,307.

WO 94/18992, published Sep. 1, 1994.

Bischoff J R. et al., “An Adenovirus Mutant that Replicates Selectivelyin p53-Deficient Human Tumor”, Science 274(52861:373-376 (1996).

Bos, J, “ras oncogenes in human cancer: a review”, Cancer Res.49:4682-4689 (1989).

Campbell, S. L. et al., “Increasing complexity of Ras signaling”,Oncogene 17: 1395-1413 (1998).

Chandron and Nibert. “Protease cleavage of reovirus capsid protein mu1and mu1C is blocked by alkyl sulfate detergents, yielding a new type ofinfectious subvirion particle”, J. of Virology 72(1):467-75 (1998).

Chang et al., J. Virol. 69:6605-6608 (1995).

Chang et al., Proc. Natl. Acad. Sci. 89:4825-4829 (1992).

Chang et al., Virol. 194:537-547 (1993).

Fueyo, J., et al., “A Mutant Oncolytic Adenovirus Targeting the RbPathway Produces Anti Glioma Effect in Vivo”, Oncogene 19(1):2-12(2000).

Gutkind, J. S., “The pathways connecting G protein-coupled receptors toWe nucleus through divergent mitogen-activated protein kinase cascades”.J Biol Chem. 273:1839-1842 (1998).

Kawagishi-Kobayashi, M. et al., Mol. Cell. Biol. 17:4146-4158 (1997).

Nemunaitis, J., “Oncolytic viruses”, J. Invest. New Drugs 17:375-386(1999).

Nibert, M. I., Schiff, L. A., and Fields, B. N., “Reoviruses and theirreplication”. pages 1557-96 in Virology (Fields et al., 3rd Edition),Lippeneott-Raven Press, 1996.

Romano et al., Mol. Cell. Bio. 18(12):7304-7316 (1998).

Sharp et al., Virology 250:302-315 (1998).

Smith, R. E., et al., “Polypeptide components of virions, top componentand cores of reovirus type 3”, Virology, 39:791-800 (1969).

Smith. C. A. et al., “Correlations among p53, Her-2/neu, and rasoverexpression and aneuploidy by multiparameter flow cytometry in humanbreast cancer: evidence for a common phenotypic evolutionary pattern ininfiltrating ductal carcinomas”, Clin Cancer Res. 6(1):112-26 (2000).

All of the publications, patents and patent applications cited above orelsewhere in this application are herein incorporated by reference intheir entirety to the same extent as if the disclosure of eachindividual publication, patent application or patent was specificallyand individually indicated to be incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

With recent developments in the field of oncology and cell biology,researchers have been able to begin drug development programs thatspecifically target the underlying cause of cancer, particularly if thecause is the deficiency or mutation of specific gene products.Therefore, if clinicians have the tools to determine the cause of cancerfor each cancer patient, a treatment regime can be chosen which istailored for the specific cause with optimized efficacy.

The ras oncogene accounts for a large number of tumors. Activatingmutations of the ras gene itself occur in about 30% of all human tumors(Bos, J. L., 1989), primarily in pancreatic (90%), sporadic colorectal(50%) and lung (40%) carcinomas, as well as myeloid leukemia (30%). Inaddition to mutations of the ras gene itself, activation of the factorsupstream or downstream of ras in the ras pathway is also associated withtumors. For example, overexpression of HER2/Neu/ErbB2 or the epidermalgrowth factor (EGF) receptor is common in breast cancer (25-30%), andoverexpression of platelet-derived growth factor (PDGF) receptor or EGFreceptor is prevalent in gliomas and glioblastomas (40-50%). EGFreceptor and PDGF receptor are both known to activate ras upon bindingto their respective ligand, and v-erbB encodes a constitutivelyactivated receptor lacking the extracellular domain. Altogether, directmutation of the ras oncogene or an upstream element in the ras pathwayis believed to occur in approximately two thirds of all tumors.

Given the significant role of the ras pathway in tumorigenesis, it isdesirable to be able to determine if a tumor is associated withactivation of the ras pathway so that a specifically tailored treatmentregime may be developed. Prior to the present invention, however, therehas not been a simple and sensitive method of diagnosing the associationof a cancer with the ras pathway. While mutations in the ras structuralgene may he detected with a high sensitivity by polymerase chainreaction (PCR), there are many other factors in the ras pathway whichmay be the cause of high ras activity, such as mutations in the ras geneflanking sequences which lead to abnormally high expression level of theras gene product, mutations in the structural genes of a factor upstreamor downstream of ras in the ras pathway, or regulatory mutations whichaffect the expression levels of these upstream or downstream factors.Therefore, PCR for the ras gene does not precisely identify all cancersassociated with activation of the ras pathway. The need remains for asimple and precise method of diagnosing ras-activated tumors.

SUMMARY OF THE INVENTION

The present invention provides a method of diagnosing neoplasms havingparticular phenotypes, particularly neoplasms mediated by abnormallyhigh activity of the ras pathway, by using reovirus or other similaroncolytic viruses. Reovirus does not replicate in normal cells. However,reovirus selectively replicates in cells with an activated ras pathway,winch leads to death of these cells. The ras pathway in these cells maybe activated due to mutations of the ras structural gene orabnormalities of any other factor in the ras pathway which lead toactivation of the pathway. Therefore, a cell which becomes neoplasticdue to, at least in part, elevated ras pathway activities can bediagnosed by its susceptibility to reovirus replication.

Accordingly, one aspect of the present invention provides a method ofdetecting ras, activated neoplastic cells in a biological sample,comprising contacting the sample with a reovirus and determining theability of the reovirus to replicate in the sample, wherein the abilityof the reovirus to replicate indicates the presence of ras-activatedneoplastic cells in the sample.

The biological sample is preferably from a mammal, particularly a human.Any reovirus capable of replicating in ras-activated cells may be usedin the present invention, for example a mammalian reovirus or an avianreovirus. The mammalian reovirus is preferably a serotype 3 reovirus andmore preferably a Dearing strain reovirus.

In a preferred embodiment, the biological sample is from an animalbearing a neoplasm selected from the group consisting of lung cancer,prostate cancer, colorectal cancer, thyroid cancer, renal cancer,adrenal cancer, liver cancer, pancreatic cancer, breast cancer,hematopoietic cancer and central and peripheral nervous system cancer.

Another aspect of the present invention provides a method of diagnosinga ras-activated neoplasm in an animal, comprising:

-   -   (a) removing, a biological sample from the animal, wherein the        sample comprises cells;    -   (b) contacting the sample with a reovirus under conditions which        allow the reovirus to replicate in ras-activated cells;    -   (c) determining the ability of the reovirus to replicate in the        sample; and    -   (d) identifying the animal as having a ras-activated neoplasm if        the reovirus can replicate in the sample.

The animal is preferably a mammal, particularly a human. Any reoviruscapable of replicating in ras activated cells may be used in the presentinvention, for example a mammalian reovirus or an avian reovirus. Themammalian reovirus is preferably a serotype 3 reovirus and morepreferably a Dearing strain reovirus.

In a preferred embodiment, the biological sample is from an animalbearing a neoplasm selected from the group consisting of lung cancer,prostate cancer, colorectal cancer, thyroid cancer, renal cancer,adrenal cancer, liver cancer, pancreatic cancer, breast cancer,hematopoietic cancer and central and peripheral nervous system cancer.

Another aspect of the present invention provides a method of treating orameliorating a ras-activated neoplasm in an animal, comprising:

-   -   (a) identifying a ras-activated neoplasm in the animal by        removing a group of cells from the animal, contacting the cells        with a reovirus under conditions which allow the reovirus to        replicate in ras-activated cells, and identifying the cells as        comprising ras-activated neoplastic cells if the reovirus can        replicate in the cells; and    -   (b) administering to the animal an effective amount of a        therapeutic agent that is selective for ras-activated neoplasms.        The therapeutic agent that is selective for ras-activated        neoplasms is preferably an oncolytic virus. The oncolytic virus        is preferably a reovirus, an adenovirus mutated in the VA1        region, a vaccine virus mutated in the K3L and/or E3L region, a        parapoxvirus orf virus mutated in the OV20.0L gene, an influenza        virus mutated in the NS-1 gene, a herpes virus mutated in the        γ₁34.5 gene, a vesicular stomatitis virus (VSV), or a Newcastle        virus. Other therapeutic agents that are selective for        ras-activated neoplasms include, without being limited to,        farnesyl transferase inhibitors (FTIs) and RAF kinase        inhibitors.

In any embodiment of the present invention, the reovirus may be arecombinant reovirus. The recombinant reovirus may be generated byco-infection of mammalian cells with different subtypes of reovirus. Therecombinant reovirus may be naturally-occurring ornon-naturally-occurring. The recombinant reovirus may be front two ormore strains of reovirus, particularly two err more strains of reovirusselected from the group consisting of strain Dearing, strain Abney,strain Jones, and strain Lang. The recombinant reovirus may also resultfrom reassortment of reoviruses from different serotypes, such asselected from the group consisting of serotype 1 reovirus, serotype 2reovirus and serotype 3 reovirus. The recombinant reovirus may comprisenaturally-occurring variant coat protein coding sequences or mutatedcoat protein coding sequences.

In addition to reovirus, a number of other oncolytic viruses are alsoselective for ras-activated neoplasms, and therefore they can be used topractice the present invention in the same manner as reovirus. Theseviruses include, without being limited to, adenoviruses mutated in theVA1 region, vaccine viruses mutated in the K3L and/or L3L region,parapoxvirus orf viruses mutated in the OV20.0L gene, influenza virusesmutated in the NS-1 gene, or herpes viruses mutated in the γ₁34.5 gene.Thus, for example, one aspect of the present invention provides a methodof detecting ras-activated neoplastic cells in a biological sample,comprising contacting the sample with an oncolytic virus thatselectively replicates in PKR-deficient cells, and determining theability of the virus to replicate in the sample, wherein the ability ofthe virus to replicate indicates the presence of ras,activatedneoplastic cells in the sample. Preferably, the oncolytic virus isselected from the group consisting of adenoviruses mutated in the VA1region, vaccine viruses mutated in the K3L and/or E31. region,parapoxvirus orf viruses mutated in the OV20.01. gene, influenza virusesmutated in the NS-1 gene, and herpes viruses mutated in the γ₁34.5 gene.

Moreover, many other oncolytic viruses that are capable of selectivelyinfecting particular tumor cells are also useful in the presentinvention in the same manner as reovirus. For example, vesicularstomatitis virus (VSV) can be used to diagnose interferon-resistanttumors, the ONYX-015 virus can be used to diagnose p53-deficient virus,and Delta24 virus can be used to diagnose Rb-deficient tumors. However,the oncolytic virus useful in the present invention is preferably not anadenovirus, particularly not the ONYX-015 virus.

Further provided by the present invention are methods of treating orameliorating interferon-resistant tumors, p53-deficient tumors, orRb-deficient tumors by rust contacting a biological sample harvestedfrom a tumor with a virus selected from the group consisting of VSV.ONYX-015 and Delta24, then treating the tumor with an appropriatetherapeutic agent upon positive diagnosis.

Yet another aspect of the present invention provides a kit comprising areovirus and a means for detecting replication of the reovirus. Thedetection means can be a pair of primers specific for the nucleic acidof the reovirus, and may optionally include reagents for PCR, Thedetection means can also be an antibody specific for a reovirus protein,as well as accompanying reagents such as secondary antibodies. Thedetection means can further he slides and dyes suitable for observingthe morphology of infected cells under the microscope, or virus culturemedia and cells that can be used to determine the titer of the reovirus.Similarly, the present invention also provides kits comprising anothervirus capable of replicating in specific tumor cells, as well as meansfor detecting replication of the virus. Examples of these virusesinclude, without being limited to, VSV, ONYX-015 virus, and Delia24virus.

Another aspect of this invention provides a kit comprising at least twoviruses which can be used to phenotype minors according to the presentinvention. The viruses arc preferably selective for neoplasms withdifferent phenotypes. Preferably, the viruses are selected from thegroup consisting of reovirus, VSV, the ONYX-015 virus, and the Delta24virus.

Yet another aspect of this invention provides a kit comprising a virususeful for diagnosis of a neoplasm of a particular phenotype, as well asa therapeutic agent selective for the neoplasm.

Furthermore, since oncolytic viruses selectively replicate in neoplasticcells but not normal. cells, another aspect of the present inventionprovides a method of diagnosing the presence of a neoplasm in a mammal,comprising contacting a sample of cells from said mammal with anoncolytic virus, wherein the ability of said virus to replicate in saidsample indicates the presence of a neoplasm in said mammal.

Other aspects of the present invention would be evident in view of theentire disclosure of the present application.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of diagnosing neoplasms havingparticular phenotypes by using oncolytic viruses. In particular, tumorsmediated by abnormally high activity of the ras pathway can be diagnosedusing reovirus. Reovirus does not replicate in normal cells. However,reovirus selectively replicates in cells with an activated ras pathway,which leads to death of these cells. Therefore, a ras-activated rumorcan be diagnosed by its susceptibility to reovirus replication. Thediagnosis will then facilitate the treatment or amelioration of thetumor with greater efficiency.

This invention can further be applied to diagnose and/or treat orameliorate other tumors, such as interferon-resistant tumors,p53-deficient tumors and Rb-deficient tumors. Kits useful in thediagnosis or treatment disclosed herein are also provided.

Prior to describing the invention in further detail, the terms used inthis application are defined as follows unless otherwise indicated.

Definitions

As used herein, “neoplastic cells”, also known as “cells with aproliferative disorder”, refer to cells which proliferate without thenormal growth inhibition properties. A new growth comprising neoplasticcells is a “neoplasm” or “tumor”. A neoplasm is an abnormal tissuegrowth, generally forming a distinct mass, that grows by cellularproliferation more rapidly than normal tissue growth. Neoplasms may showpartial or total lack of structural organization and functionalcoordination with normal tissue. As used herein, a neoplasm is intendedto encompass hematopoietic neoplasms as well as solid neoplasms.

A neoplasm may be benign (benign tumor) or malignant (malignant tumor orcancer). Malignant tumors can be broadly classified into three majortypes. Malignant neoplasms arising from epithelial structures are calledcarcinomas, malignant neoplasms that originate from connective tissuessuch as muscle, cartilage, fat fir bone are called sarcomas andmalignant tumors affecting hematopoietic structures (structurespertaining to the formation of blood cells) including components of theimmune system, are called leukemias and lymphomas. Other neoplasmsinclude, but are not limited to neurofibromatosis.

A “PKR deficient cell” is a cell in which PKR is not activated as innormal cells. Such PKR deficiency may he due to, for example, a mutationin the PKR gene or a reduced level of PKR protein or activity. Forexample, ras-activated neoplastic cells are PKR deficient because theactivated ras pathway blocks phosphorylated of PKR. Assays for PKRprotein or activity levels are known in the art.

As used herein, “ras-activated neoplastic cells” or “ras-mediatedneoplastic cells” refer to cells which proliferate at an abnormally highrate due to, at least in part, activation of the ras pathway. The raspathway may be activated by way of ras gene structural mutation,elevated level of ras gene expression, elevated stability o the ras genemessage, or any mutation or other mechanism which leads to theactivation of ras or a factor or factors downstream or upstream from rasin the ras pathway, thereby increasing the ras pathway activity. Forexample, activation of EGF receptor, PDGF receptor or Sos results inactivation of the ras pathway. Ras-mediated neoplastic cells include,but are not limited to, ras-mediated cancer cells, which are cellsproliferating in a malignant manner due to activation of the raspathway.

A “ras-activated tumor” is a tumor in which the ras pathway isactivated.

An “interferon-resistant tumor” or “a tumor having the phenotype ofinterferon-resistance” is a tumor that can not be treated or amelioratedwith interferon-alpha, beta or gamma.

A “p53-deficient tumor” or “a tumor having the phenotype ofp53-deficiency” is a tumor in winch the level of the cellular tumorsuppressor p53 is lower than that in a normal cell.

An “Rb-deficient tumor” or “a minor having the phenotype ofRb-deficiency” is a tumor in which the level of the cellular tumorsuppressor Rb is lower than that in a normal cell.

An “oncolytic virus” is a virus that selectively kills neoplastic cells.Killing of the neoplastic cells can be detected by any methodestablished in the art, such as determining viable cell count,cytopathic effect, apoptosis of die neoplastic cells, synthesis of viralproteins in the neoplastic cells (e.g., by metabolic labeling, Westernanalysis of viral proteins, or reverse transcription polymerase chainreaction of viral genes necessary for replication), or reduction in sizeof a tumor.

As used herein, “reovirus” refers to any virus classified in thereovirus genus. The name reovirus (Respiratory and enteric orphan virus)is a descriptive acronym suggesting that these viruses, although notassociated with any known disease state in humans, can he isolated frontboth the respiratory and enteric tracts. The term “reovirus” refers toall viruses classified in the reovirus genus.

The human reovirus consists of three serotypes: type 1 (strain Lang orT1L), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strainAbney, T3D). The three serotypes are easily identifiable on the basis ofneutralization and hemagglutinin-inhibition assays (See, for example,Nibert et al., 1996).

The reovirus may be naturally occurring or modified. The reovirus is“naturally-occurring” when it can be isolated from a source in natureand has not been intentionally modified by humans in the laboratory. Forexample, the reovirus can be from a “held source”, that is, from a humanwho has been infected with the reovirus.

The reovirus may be modified but still capable of lyrically infecting amammalian cell having an active ras pathway. The reovirus may bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe proliferating cells. Pretreatment with a protease removes the outercoat or capsid of the virus and may increase the infectivity of thevirus. The reovirus may he coated in a liposome or micelle (Chandron andNibert, 1998) to reduce or prevent an immune response from a mammalwhich has developed immunity to the reovirus. For example, the virionmay be treated with chymotrypsin in the presence of micelle formingconcentrations of alkyl sulfate detergents to generate a new infectioussubvirion particle.

The reovirus may be a recombinant reovirus resulting from therecombination/reassortment of genomic segments from two or moregenetically distinct reoviruses. The recombinant reovirus may be fromtwo or more types of reoviruses with differing pathogenic phenotypessuch that it contains different antigenic determinants, thereby reducingor preventing an immune response by a mammal previously exposed to areovirus subtype. Recombinant reoviruses may also exhibit differentbiological activities (e.g., replication activities in neoplastic cellsand biodistribution) compared to the original reoviruses.Recombination/reassortment of reovirus genomic segments may occur innature following infection of a host organism with at least twogenetically distinct reoviruses. Recombinant virions can also begenerated in cell culture, for example, by co-infection of permissivehost cells with genetically distinct reoviruses (Nibert et al. 1996).

Accordingly, the invention contemplates the use of recombinantreoviruses resulting from reassortment of genome segments from two ormore genetically distinct reoviruses, including but not limited to,human reovirus, such as type 1 (e.g., strain Lang), type 2 (e.g., strainJones), and type 3 (e.g., strain Dearing or strain Abney), non-humanmammalian reoviruses, or avian reovirus. The invention furthercontemplates the use of recombinant reoviruses resulting fromreassortment of genome segments from two or more genetically distinctreoviruses wherein at least one parental virus is geneticallyengineered, comprises one or more chemically synthesized genomicsegment, has been treated with chemical or physical mutagens, or isitself the result of a recombination event. The invention furthercontemplates the use of recombinant reovirus that has undergonerecombination in the presence of chemical mutagens, including but notlimited to dimethyl sulfate and ethidium bromide, or physical mutagens,including but not limited to ultraviolet light and other forms ofradiation.

The invention further contemplates recombinant reoviruses that comprisedel ions or duplications in one or more genome segments, that compriseadditional genetic information as a result of recombination with a hostcell genome, or that comprise synthetic genes.

“Phenotyping” a rumor means classifying a tumor according to itsphenotype. For example, tumor phenotypes include ras pathway activation,interferon-resistance, p53-deficiency and Rb-deficiency. The phenotypesare not mutually exclusive, namely, a tumor may he phenotyped into morethan one class.

A “biological sample” is a sample collected from a biological subject,such as an animal.

An “effective amount” is an amount which is sufficient to achieve theintended purposes. For example, an effective amount of reovirus for thepurpose of treating or ameliorating a disease or medical condition is anamount sufficient to result in a reduction or complete removal of thesymptoms of a disease or medical condition. The effective amount of agiven therapeutic agent will vary with factors such as the nature of theagent, the route of administration, the size and species of the animalto receive the therapeutic agent, and the purpose of the administration.The effective amount in each individual case may be determinedempirically by a skilled artisan according to established methods inthe. art.

“Treating or ameliorating” a disease or medical condition means thereduction or complete removal of the symptoms of a disease or medicalcondition.

A therapeutic agent is “selective” for a particular disease or medicalcondition if the agent is more effective for the disease or medicalcondition than for other diseases or medical conditions. Similarly, atherapeutic agent is selective for a particular group of neoplasticcells if the agent kills the particular group of neoplastic cells withhigher efficiency than other neoplastic cells.

Method

The present invention is useful in the precise phenotyping, of tumors,thereby facilitating the development of a treatment regime that istailored for a specific tumor. In a preferred embodiment, reovirus isused to infect a biological sample harvested from a tumor-bearinganimal. Since reoviruses selectively infect ras-activated neoplasticcells but not normal cells or tumor cells in which the ras pathway isnot activated, the present method enables the practitioner to preciselydetermine if the tumor is associated with ras pathway activation. Ifdiagnosed to be ras activated, the tumor can then be treated withras-specific treatment regimens, such as reovirus therapy (U.S. Pat. No.6,136,307)

The ras pathway is a complex signal transduction pathway that leads tocellular proliferation. Ras is a central relay in this pathway,receiving signals from upstream elements (e.g., growth factor receptors)and transmitting them to downstream elements.

Many growth factor receptors such as epidermal growth factor (EGF)receptor, platelet-derived growth factor (PDGF) receptor, as well as EGFreceptor-related molecules (e.g. Her-2/Neu/ErbB2), possess an intrinsictyrosine kinase activity which is activated by ligand-induced receptordimerization. This results in autophosphorylation of the receptor ontyrosine residues and the binding of proteins containing Src-homology 2(SH2) domains. Two such SH2 proteins are Grb2 and SHC which indirectlyactivate the plasma membrane-associated, small GTP-binding protein Ras.Ras activation also occurs in response to ligand binding to seventransmembrane domain G-protein coupled receptors (e.g. Gutkind. 1998).Activation of Ras and other growth factor receptor-regulated signalingpathways ultimately leads to changes in the cytoskeleton and geneexpression which are necessary for cellular proliferation,differentiation, and transformation (reviewed in Campbell et al., 1998).

The three human ras genes (Ha-Ras, N-Ras, and Ki-Ras) encode 4 proteins(due u alternative splicing of the Ki-Ras mRNA). Under normalcircumstances, Ras proteins cycle between an active (GTP-bound) stateand an inactive (GDP-bound) state. Ras activation occurs by exchange ofhound GDP for GTP, which is facilitated by a family of guaninenucleotide exchange factors. Ras inactivation occurs by hydrolysis ofbound GTP m GDP. This reaction is facilitated by GTPase activatingproteins (GAPs). In many human cancers. Ras proteins becomeoncogenically activated by mutations which destroy their GTPaseactivity, and thus deregulate Ras signaling (reviewed in Campbell etal., 1998).

Multiple candidate Ras effectors exist that may serve downstream of Rasin signal transduction and oncogenic transformation, including membersof the Rho family of small GTPases, phosphatidylinositol-3 kinase (PI3K)and the serine/threonine protein kinase c-Raf-1 (reviewed in Campbell etal., 1998). Raf-mediated signaling is the best characterized Raseffector pathway. Activated Ras recruits Raf to the membrane where Rafactivation occurs. Activated Raf is the initial component of a kinasecascade. the Mitogen-Activated Protein Kinase (MAPK) cascade. Rafphosphorylates and activates the MEK1 and MEK2 (MAPK/ERK kinase) proteinkinases which, in turn, phosphorylate and activate the Extracellularsignal Regulated Kinases ERK1 and ERK2 (also known as MAPK1 and MAPK2).Unlike their downstream targets, ERK1,2, the MEK1,2 proteins are highlyspecific enzymes whose only known substrates are the ERK1,2 proteins.Upon activation, ERK1 and ERK2 phosphorylate (and thus regulate) avariety of target proteins, including nuclear transcription factors,leading to the ultimate cellular response.

Accordingly, numerous events can lead to activation of the ras pathway.For example, a mutation may occur in any of the three ras structuralgenes. Structural mutations may also take place in the receptorsupstream of ras, the signal transducers downstream from ras (such as rafor mek1,2), or the ultimate effectors MAPK1 and 2. Similarly, regulatorymutations that lead to abnormally high levels of expression of anyprotein in the ras pathway may also cause mitogenic cellular responses.Such regulatory mutations may occur anywhere in the regulatory sequencesof a ras pathway member, or even in the structural or regulatory regionof a factor that controls the expression of a ras pathway member.Consequently, detection of aberration of any specific member in the raspathway is not an efficient way to determine if the ras pathway isactivated.

It is possible to measure the activity of MAPK, the ultimate effector ofthe ras pathway, since constitutive activation of MAPK is indicative ofras pathway activation. However, such a biochemical approach requires asubstantial amount of sample material, as well as tedious proceduressuch as extraction and/or partial purification of MAPK.

By detecting the ras activated phenotype rather than aberration of anyspecific gene or gene product, the present invention is useful whetherthe ras pathway activation is due to mutation of the ras structuralgene, regulatory sequences of the ras gene. or any other factor in theras pathway. Furthermore, the present method is relatively simple,without the need to extract or purity an enzyme from the sample.

The ability of reovirus to infect cells in a sample can he determined byany method in the art. For example, reovirus nucleic acid replicationcan he measured by polymerase chain reaction with primers specific forthe reovirus used: reovirus protein synthesis can be detected byspecific antibodies; infected cells can be observed under a microscopeand evidence of cytopathic effects induced by the reovirus detected; andreplicated reovirus can be harvested from the sample, and virus titerdetermined, to assess if viral replication has taken place. Othermethods of determining the presence of reovirus replication are known toor may be developed by people of ordinary skill in the art.

It should he noted that a tumor may contain multiple oncogenicabnormalities. In particular, it has been reported that ras activationis often preceded by p53 over-expression in breast cancer (Smith et al.,2000). The presence of other oncogenic abnormalities in addition to raspathway activation, however, does not impede the ability of a therapyregime specifically tailored for ras-activated tumors. For example,reovirus can still selectively kill ras-activated neoplastic cells evenif the cells also contain abnormally high levels or p53.

Furthermore, since reovirus selectively replicates in ras-activatedneoplastic cells but not normal cells, another aspect of the presentinvention provides a method of diagnosing the presence of a neoplasm ina mammal, comprising contacting a sample of cells from said mammal witha reovirus under conditions that allow the. reovirus to replicate inras-activated cells, wherein the ability of said reovirus to replicatein said sample indicates the presence of a neoplasm in said mammal.

Similar to reovirus, a number of other oncolytic viruses alsoselectively replicate in ras-activated cells. It is contemplated thatthese oncolytic viruses can be employed to practice the presentinvention in the same manner as reovirus. These viruses typically arcmutants that are sensitive to the double stranded RNA kinase (PKR),whereas their wild type counterparts are not sensitive to PKR.

Normally, when a virus enters a cell, PKR is activated and blocksprotein synthesis, and the virus can not replicate in this cell. Someviruses have developed a system to inhibit PKR and facilitate viralprotein synthesis as well as viral replication. For example, adenovirusmakes a large amount of a small RNA, VA1 RNA. VA1 RNA has extensivesecondary structures and hinds to PKR in competition with the doublestranded RNA (dsRNA) which normally activates PKR. Since it requires aminimum length of dsRNA to activate PKR, VA1 RNA does not activate PKR.Instead, it sequesters PKR by virtue of its large amount. Consequently,protein synthesis is not blocked and adenovirus can replicate in thecell.

Ras-activated neoplastic cells are not subject to protein synthesisinhibition by PKR, because ras inactivates PKR. These cells aretherefore susceptible to viral infection even if the virus does not havea PKR inhibitory system. Accordingly, if the PKR inhibitors inadenovirus is mutated so as not to block PKR function anymore, theresulting virus does not infect normal cells due to protein synthesisinhibition by PKR, but they replicate in ras-activated neoplastic cellswhich lack PKR activities.

Accordingly, a virus that is modified or mutated such that it does notinhibit PKR function selectively replicates in ras-activated neoplasticcells while normal cells are resistant. Preferably. the virus is anadenovirus mutated in the VA1 region, a vaccinia virus mutated in theK3L, and/or E3L region, a parapoxvirus orf virus mutated in the OV20.0Lgene, an influenza virus mutated in the NS 1 gene, or a herpes virusmutated in the γ₁34.5 gene.

The viruses can be modified or mutated according to the knownstructure-function relationship of the viral PKR inhibitors. Forexample, since the amino terminal region of E3 protein interacts withthe carboxy-terminal region domain of PKR, deletion or point mutation ofthis domain prevents anti-PKR function (Chang et al., 1992, 1993, 1995;Sharp et al., 1998; Romano et al., 1998). The K3L gene of vaccinia virusencodes pK3, a pseudosubstrate of PKR. There is a loss-of-functionmutation within K3L. Truncations or point mutations within theC-terminal portion of K3L protein that is homologous to residues 79 to83 in eIF 2 abolish PKR inhibitory activity (Kawagishi-Kobayashi et al.,1997),

In another embodiment of the present invention, the vesicular stomatitisvirus (VSV) can he used to diagnose interferon-resistant tumors.Interferons are circulating factors which bind to cell surface receptorsand ultimately lead to both an antiviral response and au induction ofgrowth inhibitory and/or apoptotic signals in the target cells. Althoughinterferons can theoretically be used to inhibit proliferation of tumorcells, this attempt has not been very successful because ofminor-specific mutations of members of the interferon pathway.

However, by disrupting the interferon pathway to avoid growth inhibitionexerted by interferon, tumor cells may simultaneously compromise theiranti-viral response. Indeed, it has been shown that VSV, an enveloped,negative-sense RNA virus, rapidly replicated in and killed a variety ofhuman tumor cell lines in the presence of interferon, while normal humanprimary cell cultures were apparently protected by interferon. VSV canthus be used to diagnose interferon-resistant yet VSV-sensitive tumors.Like the reovirus embodiment, VSV-based diagnosis is an assessment ofthe phenotype and does not depend on the mechanism of interferonresistance.

In another embodiment of the present invention, the ONYX-015 virus canbe used to diagnose p53-deficient tumors. p53 is a potent tumorsuppressor, which is present in every cell and controls cell growth.Since viruses rely on the cellular proliferation machinery to replicate,they are subject to p53 regulation and can not over-replicate. Certainadenovirus, SV40 and human papilloma virus, however, include proteinswhich inactivate p53, thereby allowing their own replication (Nemunaitis1999).

For adenovirus serotype 5 this protein is a 55 Kd protein encoded by theE1B region. It the FIB region encoding this 55 kd protein is deleted, asin the ONYX-015 virus (Bischoff et al, 1996; WO 94/18992), the 55 kd p53inhibitor is no longer present. As a result, when ONYX-015 enters anormal cell, p53 functions to suppress cell proliferation as well asviral replication. Therefore, ONYX-015 does not replicate in normalcells. On the other hand, in neoplastic cells with disrupted p53function, ONYX-015 can replicate and eventually cause the cell to die.Accordingly, this virus can be used to detect p53-deficient neoplasticcells in a sample. A person of ordinary skill in the art can also mutateand disrupt the p53 inhibitor gene in adenovirus 5 or other virusesusing established techniques, and the resulting viruses are useful inthe present method to diagnose p53-deficient tumors.

Similarly, the Delta24 virus can be used to diagnose Rb-deficientrumors. The Delta24 virus is a mutant adenovirus carrying a 24 base pairdeletion in the E1A region (Fueyo et al., 2000). This region isresponsible for binding to the cellular tumor suppressor Rb andinhibiting Rb function, thereby allowing the cellular proliferativemachinery, and hence virus replication, to proceed in an uncontrolledfashion. Delta24 has a deletion in the Rb binding, region and does notbind to Rb. Therefore, replication of the mutant virus is inhibited byRb in a normal cell. However, if Rb is inactivated and the cell becomesneoplastic, Delta24 is no longer inhibited. Instead, the mutant virusreplicates efficiently and lyses the Rb-deficient cell. Accordingly, theDelta24 virus can be used to determine if a sample contains Rb-deficienttumor cells.

As is the case with the ras-activated tumor cells, p53-deficient orRb-deficient cells may be the result of a variety of reasons. Forexample, a mutation in the structural gene of p53 or Rb may lead to amalfunctioning gene product or poor translation, a imitation in theregulatory sequence of the p53 or Rb gene may cause reduced amount oftranscription, a mutation in a transcription factor for the p53 or Rbgene may result in deficient p53 or Rb production, or a mutation in aco-factor necessary for p53 or Rb function may also he the reason ofp53- or Rb-deficiency. Since the present invention detects thephenotype, rather than structural aberration of the p53 or Rbgene/protein only, it is more powerful than structure-based methods,such as PCR.

Once the phenotype of a tumor has been determined, the tumor can hetreated according to its phenotype. For example, a ras-activated tumorcan be treated by reovirus, or inhibitors of the ras pathway.Accordingly, the present invention also provides a method of treating orameliorating a ras-activated neoplasm in an animal, comprisingidentifying a ras-activated neoplasm in the animal by removing a groupof cells from the animal, contacting the cells with a reovirus underconditions which allow the reovirus to replicate in ras-activated cells,identifying the cells as comprising ras-activated neoplastic cells ifthe reovirus can replicate in the cells, and administering an effectiveamount of reovirus to the mammal. Reovirus therapy has been disclosed,for example, in U.S. Pat. No. 6,136,307.

Furthermore, the present invention also provides methods of treating, orameliorating a tumor, comprising collecting a sample, identifying thephenotype of the sample with VSV, he Delta24 or ONYX-015 virus, andadministering an effective amount of a suitable therapeutic agentaccording to the phenotype. The therapeutic agent may he the virusitself, or, in the case of p53 or Rb-deficiency, activators of p53 or Rbfunctions. It should be noted that Delta24 and ONYX-015 are merelyexamples to elucidate the application of the present invention, while aperson with ordinary skill in the art will be able to identify ordevelop other viruses useful in the diagnosis and treatment of tumorsaccording to the present disclosure.

As with reovirus, the use of immunoprotected or reassortant viruses ofother oncolytic viruses are also encompassed in the present invention.Furthermore, in addition to the viruses specifically discussed in thepresent application, a person of ordinary skill in the art can practicethe present invention using additional oncolytic viruses according tothe disclosure herein and knowledge available in the art. The oncolyticvirus may he a member in the family Of myoviridae, siphoviridae,podoviridae, teciviridae, corticoviridae, plasmaviridae,lipothrixviridae, fuselloviridae, poxviridae, iridoviridac,phycodnaviridae, baculoviridae, herpesviridae, adenoviridae,papovaviridae, polydnaviridae, inoviridae. microviridae, geminiviridae,circoviridae, parvoviridae, hepadnaviridae, retroviridae, cycloviridae,rcoviridae, birnaviridae, paramvxoviridae, rhabdoviridae,orthornyxoviritlac, hunyaviridae, arenaviridae, leviviridae,pieornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae,astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae,glaviviridae, togaviridae, or barnaviridae.

The present invention can be applied to any animal, particularlymammals. Preferred mammals include dogs, cats, sheep, goats, cattle,horses, pigs, humans and non-human primates. Most preferably, the mammalis human.

Kits

The present invention provides kits useful for the diagnosis and/ortreatment of tumors. One aspect of the present invention provides a kitcomprising a reovirus and a means for detecting replication of thereovirus. The detection means can be a pair of printers specific for thenucleic acid of the reovirus, and may optionally include reagents forPCR. The detection means can also be an antibody specific for a reovirusprotein, and may optionally contain the accompanying reagents such assecondary antibodies. The detection means can further be slides and dyessuitable for observing the morphology of infected cells under themicroscope, or virus culture media and cells that can be used todetermine the titer of the reovirus. Similarly, the present inventionalso provides kits comprising another virus capable of replicating inspecific tumor cells, as well as means for detecting replication of thevirus. Examples of these viruses include, without being limited to, VSV,the ONYX-015, and Delta24 virus.

Another aspect of this invention provides a kit comprising at least twoviruses which can be used to phenotype tumors according to the presentinvention. Preferably, the viruses arc selected from the groupconsisting of reovirus, VSV, the ONYX-015 virus, and the Delta24 virus.

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of the presentinvention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. Abbreviations not defined have their generally acceptedmeanings.

° C.=degree Celsius

hr=hour

min=minute

μM=micromolar

mM=millimolar

M=molar

ml=milliliter

μl=microliter

mg=milligram

μg=microgram

PAGE=polyacrylamide gel electrophoresis

rpm=revolutions per minute

FBS=fetal bovine serum

DTT=dithiothrietol

SDS=sodium dodecyl sulfate

PBS=phosphate buffered saline

DMEM=Dulbeceo's modified Eagle's medium

α-MEM=α-modified Eagle's medium

β-ME=β-mercaptoethanol

MOI=multiplicity of infection

PFU=plaque forming units

EGF=epidermal growth factor

PDGF=platelet derived growth factor

CPE=cytopathic effect

VSV=vesicular stomatitis virus

PCR=polymerase chain reaction

SH2=src-homology 2

Example 1 Phenotyping a Tumor with Reovirus

A lump is found in a 65 year old woman when she has her regularmammogram. A sample is collected from the lump during biopsy and appearsto be a malignant tumor. In order to determine if the tumor containsras-activated cells, the sample is placed in cell culture and incubatedwith reovints.

The Dearing strain of reovirus scrotype 3 is propagated in suspensioncultures of L-929 cells purified according to Smith (Smith et al., 1969)with the exception that β-mercaptoethanol (β-ME) is omitted from theextraction buffer. The panicle/PFU ratio for purified reovirus istypically 100/1. The biopsy sample is minced in DMEM, incubated withreovirus for 2 hours at 37° C., changed to fresh DMEM plus 20% EBS, andcultured for 48 hours. Thereafter, the supernatant of the culture iscollected and reovirus titer is determined. The result indicates thatreovirus has replicated in the sample. Therefore, the breast tumorcontains ras-activated tumor cells.

1.-33. (canceled)
 34. A kit comprising at least two different oncolyticviruses, wherein each oncolytic virus selectively replicates in one ormore of ras-activated neoplastic cells, interferon resistant neoplasticcells, p53-deficient neoplastic cells or Rb-deficient neoplastic cells.35. The kit of claim 34, wherein the oncolytic viruses are selected fromthe group consisting of p53 expressing viruses, reovirus, vesicularstomatitis virus (VSV), ONYX-15, Delta24, adenoviruses mutated in theVA1 region, vaccinia viruses mutated in the K3L or E3L region,parapoxvirus orf viruses mutated in the OV20.0L gene, and herpes virusesmutated in the γ₁34.5 gene.
 36. The kit of claim 34, wherein theoncolytic viruses are selected from the group consisting of reovirus,VSV, ONYX-15 and Delta24.
 37. The kit of claim 35, wherein the reovirusis a mammalian reovirus.
 38. The kit of claim 37, wherein the mammalianreovirus is a serotype 3 reovirus.
 39. The kit of claim 38, wherein theserotype 3 reovirus is a Dearing strain reovirus.
 40. A kit comprisingat least two different oncolytic viruses, wherein each oncolytic virushas a different phenotype specificity selected from the group consistingof ras-pathway activation, interferon-resistance, p53 deficiency andRb-deficiency.
 41. The kit of claim 40, wherein the oncolytic virusesare selected from the group consisting of p53 expressing viruses,reovirus, vesicular stomatitis virus (VSV), ONYX-15, Delta24,adenoviruses mutated in the VA1 region, vaccinia viruses mutated in theK3L or E3L region, parapoxvirus orf viruses mutated in the OV20.0L gene,and herpes viruses mutated in the γ₁34.5 gene.
 42. The kit of claim 40,wherein the oncolytic viruses are selected from the group consisting ofreovirus, VSV, ONYX-15 and Delta24.
 43. The kit of claim 41, wherein thereovirus is a mammalian reovirus.
 44. The kit of claim 43, wherein themammalian reovirus is a serotype 3 reovirus.
 45. The kit of claim 44,wherein the serotype 3 reovirus is a Dearing strain reovirus.